Abstract 6744: Why the analytical development of agonistic antibodies is challenging? A comprehensive strategy to unlock their characterization for immunotherapy and autoimmunity

Abstract Checkpoint blockade antibodies, or checkpoint inhibitors, are established as effective cancer therapeutics. A contrasting class of therapeutics, agonistic checkpoint antibodies, has recently emerged for attenuating inflammation in autoimmune diseases. Despite showing clinical promise, their development remains challenging due to limitations in conventional checkpoint assay designs that often fail to replicate physiologically relevant conditions in vitro and are sometimes confined to assessing only inhibitory responses. Here, we introduce a novel checkpoint assay design in a co-culture format involving Fcγ receptor-expressing cells, directly measuring the stimulatory activity of agonistic antibodies. Through testing various classes of Fcγ receptors and diverse cell backgrounds, we have established a specific and robust assay design for several key checkpoint receptors such as CD40, CD137, PD-1, and BTLA. Notably, we have identified previously unrecognized agonistic activity in a clinically approved blockade antibody, underscoring the importance of exploring similar activity in other therapeutics of this nature. The assay is optimized as a thaw-and-use format, exhibiting high reproducibility with ease of execution, and qualified for potency and stability studies as needed for commercial release testing in a quality-controlled environment. Citation Format: Gaurav Agrawal, Venkatesh Chari, Luhan Yang, Jennifer Lin-Jones, Alpana Prasad, Jane Lamerdin. Why the analytical development of agonistic antibodies is challenging? A comprehensive strategy to unlock their characterization for immunotherapy and autoimmunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6744.